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apoptosis upon over-expression with pcDNA6-cDNA - how prove sequence specificit


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#1 greenhorn

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Posted 22 June 2011 - 01:26 PM

Hello everyone,

I am trying to establish a cell line stably over-expressing my gene of interest. Transfecting the cDNA in a pcDNA6-plasmid lead to apoptosis of the cells with a 3 day delay compared to untransfected controls. Is there any good way to demonstrate clearly that this is sequence specific and definitely not related to transfection efficiency? Or a first step before I'd start cloning my cDNA into other (i.e. inducible) systems or similiar?

Any help would be really appreciated!!

#2 bob1

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Posted 22 June 2011 - 06:08 PM

Did you do an "empty" vector control (i.e. pcDNA6 with no insert) and a non-target vector control (i.e. pcDNA6 with another cDNA or a scrambled version of your cDNA)?

#3 greenhorn

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Posted 22 June 2011 - 09:07 PM

Did you do an "empty" vector control (i.e. pcDNA6 with no insert) and a non-target vector control (i.e. pcDNA6 with another cDNA or a scrambled version of your cDNA)?


Hello,

Yes, I had the empty vector, which was proliferating happily under selection but no scrambled cDNA. How could I make a scrambled version of my cDNA?

Thanks so much for any help!

#4 bob1

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Posted 23 June 2011 - 05:37 PM

You can use another gene as a "non-target" control - if the cells die when you transfect this in, then it is a transfection issue, not a property of your gene.

You should also check expression of your gene in the cells by western and/or RT-PCR.

#5 greenhorn

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Posted 25 June 2011 - 10:58 AM

Thank you very much! I will measure mRNA expression and transfect another gene in parallel as a first step :o).




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